The plasminogen activator inhibitor-1 (PAI-1) is one of the serine protease inhibitors produced from the vascular endothelium or like. PAI-1 binds to a plasminogen activator (PA), which is a serine protease, to inactivate an enzymatic activity of PA (Mol. Cell. Endocrinol., 1990, Vol. 68, p. 1-19). With respect to PAI-1, there are active PAI-1, latent PAI-1, and PAI-1 bound to and complexed with PA, and the property of PAI-1 to inhibit PA is possessed only by active PAI-1 (Blood, 1987, Vol. 70, p. 1090-1098). There are two types of PA: tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA, also referred to as urokinase), both of which activate plasminogen to convert into plasmin, thereby catalyzing a reaction that lyses fibrin produced during blood clotting (hereinafter referred to as a fibrinolytic system). Active PAI-1 binds to tPA and uPA to form a complex. Biological properties that deactivate PA have been fully elucidated, and binding of active PAI-1 to PA results in inhibition of a fibrinolytic system to thereby facilitate thrombus formation.
The characteristic of the steric structure of active PAI-1 is that the active center loop which is a binding site to PA is extended and exposed to the outside of the protein (J. Biol. Chem., 2001, Vol. 276, p. 44912-44918). In circulating blood, active PAI-1 can bind to its cofactor vitronectin to form a complex (J. Biol. Chem., 1988, Vol. 263, p. 15454-15461). Vitronectin inhibits the conformational change of an active form of PAI-1 into a latent form thereof (J. Biol. Chem., 1990, Vol. 265, p. 18490-18498). Latent PAI-1 has a stable steric structure where the active center loop is folded inward. Once PAI-1 become a latent form, it becomes incapable of binding to PA (J. Biol. Chem., 2008, Vol. 283, p. 18147-18157. Nature, 1992, Vol. 355, p. 270-273). Moreover, since PAI-1 in complex with tPA or uPA is each bound to tPA or uPA, this PAI-1 cannot newly bind to another PA. That is, with respect to active PAI-1, there are active PAI-1 which is present as a monomer, and active PAI-1 which is present as a complex with vitronectin, and these active forms of PAI-1 have a function to inactivate PA by binding to PA.
An increase in active PAI-1 in the plasma has been suggested to be involved in tissue fibrosis and thrombus formation through the inhibition of PA, and is consequently believed to be involved in diseases, such as pulmonary fibrosis such as idiopathic pulmonary fibrosis, interstitial pneumonia, systemic lupus erythematosus, scleroderma, diabetic nephropathy, lupus nephritis, graft-versus-host disease, glomerulonephritis, nephrotic syndrome, renal fibrosis, chronic obstructive pulmonary disease, acute kidney injury, acute lung injury, acute respiratory failure, age-related macular degeneration, disseminated intravascular coagulation, post-surgical adhesion, symptomatic vitreomacular adhesion, diabetic retinopathy, arteriosclerosis, myocardial infarction, cerebral infarction, pulmonary infarction, ocular fibrosis-accompanying disease such as conjunctival scarring after glaucoma surgery, peritoneal sclerosis, and the like.
In association with pulmonary fibrosis, it has been reported that the expression and secretion of PAI-1 are increased in lung biopsy-derived fibroblasts of idiopathic pulmonary fibrosis patients (J. Biol. Chem., 2010, Vol. 285, No. 11, p. 8196-8206). In addition, it has been confirmed in tests using pathological model mice of bleomycin-induced pulmonary fibrosis that fibrosis of the lung is inhibited in mice with a genetic deficiency of PAI-1 (J. Clin. Invest., 1996, Vol. 97, No. 1, p. 232-237). Moreover, it has also been confirmed that fibrosis of the lung is inhibited by the inhibition of PAI-1, in the cases where a low molecular weight PAI-1 inhibitor and PAI-1 siRNA were administered to the above-mentioned pathological model mice (Arterioscler. Thromb. Vase. Biol., 2008, Vol. 28, p. 672-677. Thorax, 2010, Vol. 65, p. 334-340).
Therefore, the development of a monoclonal antibody having an activity capable of inhibiting the action by active PAI-1 through specific binding to active PAI-1 is expected to be useful in the prevention and treatment of various diseases where active PAI-1 is involved in the pathogenesis thereof.
As antibodies exhibiting a functional inhibitory action on active human PAI-1, there have been reported mouse monoclonal antibodies MA-33B8 (Patent Document 1), MA-33H1F7 (Non-Patent Document 1), MA-55F4C12 (Non-Patent Document 1), and MA-56A7C10 (Patent Document 2), and a humanized antibody CT140 of MA-33B8 (Patent Document 1), and a fully human antibody MEDI-579 (also referred to as CAT-1001 or PICK167_A01-fgl IgG1, Patent Document 3) which was prepared by using a phage. Among them, MA-56A7C10 and MEDI-579 exhibit about 4.0-fold and about 20-fold higher binding activity, respectively, for active human PAI-1 than for human PAI-1 having a steric structure other than the active form (Patent Documents 2 and 3). In addition, MEDI-579 also exhibits an inhibitory effect on mouse PAI-1, and is therefore expected to have a medicinal effect on lupus nephritis, scleroderma, diabetic nephropathy and thrombosis based on the experimental results using pathological model mice of those diseases (Patent Document 3).
In the case of using an anti-human PAI-1 antibody as an antibody drug, main factors defining an effective dose of the antibody may include an inhibitory activity of the antibody on the binding of active PAI-1 and PA, selectivity of the antibody for active PAI-1, and amounts of active PAI-1 and PAI-1 of other steric structures present in the body. As used herein, the term “PAI-1 of other steric structures” refers to latent PAI-1, and PAI-1 in complex with tPA or uPA.
More than 90% of total PAI-1 in human plasma is stored in platelets. About two thirds of total PAI-1 is an active form in the plasma except for platelets (Br. J. Haematol., 1988, Vol. 70, p. 327-333. Blood, 1988, Vol. 71, p. 220-225). Activity of PAI-1 in the plasma (activity of active PAI-1 expressed in terms of binding ability to tPA) as well as amounts of total PAI-1 in the plasma (total amounts of PAI-1 detected by an anti-PAI-1 antibody that detects all of PAI-1) and amounts of a complex of PAI-1 and tPA in the plasma (amounts of a complex that is detected as a combination of an anti-PAI-1 antibody and an anti-tPA antibody) are increased in various human pathologies (Thromb. Res., 2008, Vol. 122, p. 466-472. Arterioscler. Thromb. Vasc. Biol., 2000, Vol. 20, p. 2019-2023. Nat. Med., 1996, Vol. 2, p. 800-803). Also in the plasma except for platelets, the tPA-PAI-1 complex is present as about a third of the amount of active PAI-1 (Blood, 1990, Vol. 76, p. 930-937). uPA and uPA-PAI-1 complexes as well as tPA are present in tissues. In conclusion, PAI-1 other than active PAI-1 is present in the human body in an unignorable proportion in terms of total PAI-1 amount.